User:Roberta Diaz Jimenez/Notebook/CHEM 472/2016/04/06

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Objective

Perform fourth agar diffusion disc assay, this time soaking discs into samples first and then loading them on the plates.

Materials

  1. Mueller Hinton Agar self-prepared on March 2 and stored in fridge
  2. Whatman Membrane Filter Paper (6mm discs punched out from larger discs) autoclaved in dry cycle
  3. Triangular spreading tool also autoclaved
  4. Same stock of 20mL silver and gold protein nanoparticles synthesized on March 15
  5. Specific amounts and reactions detailed at the link SRB Lab Notebook Data for this entry's date

Procedure

  • Dilutions of Protein Nanoparticle Samples following the shown dilution factors:
Concentration (x)
1
0.5
0.25
0.125
0.0625
  • Ampicillin Control Dilutions following the table shown below:
Concentration (mg/L) Concentration (ug/uL) Volume (ul) Mass (ug)
40.004250.1
80.008250.2
160.016250.4
320.032250.8
640.064251.6
  • Disc Diffusion
  1. Thaw E. Coli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 min over ice
  2. Dilute E. Coli thawed stock to OD600 (absorbance at 600nm) of 0.08-0.1 (specifically 0.1A)
  3. Plate 100µl sample of diluted thawed E. Coli solution onto Mueller Hinton agar plates by first pipetting the volume and then using a triangular spreader in circles
  4. Soak individual 6mm Whatman membrane filter paper discs directly in sample for 30 mins and load onto plates with tweezers
  5. Place discs on plate, labeled so that dilution 1, is in a particular 5th of a plate (5 dilutions total) and immediately cover plate
  6. Incubate at 37˚C overnight in incubator and check results after 24 hours of incubation
  7. After 24hrs incubation at 37˚C remove discs from incubator and measure diameters of bacterial inhibition around the discs. Results noted and discs photographed for later analysis.
  • Note: on March 30, loaded 10ul onto the membrane filter paper discs which resulted in significantly decreased inhibition very likely due to such low volume. Soaking directly in sample for 30mins and note if there is inhibition; this would allow for a method that prevents any leaking to occur once the discs are placed on the plate.
  • Note: that this technique was conducted on the benchtop. Between each loading of a disc on a plate, the tweezers used were washed with EtOH to minimize contamination. However, the plates were open to the air for a period of time during plating and loading of discs. Because we are testing for bacterial inhibition by the nanoparticles and control, if there is contamination, but inhibition we may never know that there was contamination in the first place. Discussed with Dr. Hartings and protocol was approved.

Results

  • Note: Agar disc diffusion specific dilution data, results and pictures can be found at the link SRB Lab Notebook Data for this entry's date


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